Bioactive Immune Components of Anti-Diarrheagenic Enterotoxigenic Escherichia coli Hyperimmune Bovine Colostrum Products

Abstract

Diarrhea is a common illness among travelers to resource-limited countries, the most prevalent attributable agent being enterotoxigenic Escherichia coli (ETEC). At this time, there are no vaccines licensed specifically for the prevention of ETEC-induced traveler's diarrhea (TD), and this has propelled investigation of alternative preventive methods. Colostrum, the first milk expressed after birthing, is rich in immunoglobulins and innate immune components for protection of newborns against infectious agents. Hyperimmune bovine colostrum (HBC) produced by immunization of cows during gestation (and containing high levels of specific antibodies) is a practical and effective prophylactic tool against gastrointestinal illnesses. A commercial HBC product, Travelan, is available for prevention of ETEC-induced diarrhea. Despite its demonstrated clinical efficacy, the underlying immune components and antimicrobial activity that contribute to protection remain undefined. We investigated innate and adaptive immune components of several commercial HBC products formulated to reduce the risk of ETEC-induced diarrhea, including Travelan and IMM-124E, a newer product that has broader gastrointestinal health benefits. The immune components measured included total and ETEC-specific IgG, total IgA, cytokines, growth factors, and lactoferrin. HBC products contained high levels of IgG specific for multiple ETEC antigens, including O-polysaccharide 78 and colonization factor antigen I (CFA/I) present in the administered vaccines. Antimicrobial activity was measured in vitro using novel functional assays. HBC greatly reduced ETEC motility in soft agar and exhibited bactericidal activity in the presence of complement. We have identified immune components and antimicrobial activity potentially involved in the prevention of ETEC infection by HBC in vivo.

Keywords: bactericidal activity; enterotoxigenic E. coli; functional antibodies; hyperimmune colostrum.

FIG 1

Innate immune components in bovine colostrum products (BCP). Growth hormone (GH) (A), insulin growth factor 1 (IGF-1) (B), tumor necrosis factor alpha (TNF-α) (C), and lactoferrin (D) were measured by ELISA in BCP resuspended in PBS-Tween (at a final concentration of 16 mg/ml) as described in Materials and Methods. Samples tested included ProMilk powders and tablets, IMM-124E 1-9 individual powders, IMM-124E powder and tablets, and Travelan (Table 2). Data represent the mean of duplicates when a single lot was tested (ProMilk or blended IMM-124E powder) or mean plus standard deviation (SD) (error bar) when multiple lots were available (IMM-124E HBC 1-9 [representing nine different lots of HBC powder] and two batches each of ProMilk, IMM-124E, and Travelan tablets), as listed in Table 2. Values that are statistically significantly different (P < 0.05) between HBC and ProMilk tablets by two-sample two-sided t test are indicated by an asterisk.

FIG 2

Immunoglobulin composition of BCP. Total IgG (A), IgG1 (B), IgG2 (C), and IgA (D) were measured by ELISA as described in Materials and Methods. Samples tested included ProMilk, IMM-124E, and Travelan (Table 2). Data represent Ig class or subclass content as mean of duplicates for single-lot samples or mean of multiple lots plus SD (where applicable).

FIG 3

Antibody titers specific to ETEC vaccine antigens in BCP. Specific IgG antibodies to vaccine antigens O6 LPS (A), O78 COPS (B), CFA/I (C), CFA/II (D), CS3 (E), CS4 (F), CS6 (G), and E. coli LT (H) were measured by ELISA in ProMilk, IMM-124E, and Travelan. Data represent mean ELISA units (EU) (plus SD when multiple lots were tested) per milligram of total IgG. Values that are significantly different (P < 0.05) between HBC and ProMilk tablets by two-sided two-sample t test are indicated by an asterisk.

FIG 4

Antibody titers specific to nonvaccine ETEC antigens in BCP. Specific IgG antibodies to ETEC antigens not included in the vaccine administered to the cows were analyzed. O42 LPS (A), O55 LPS (B), and O127 LPS (C) were measured by ELISA in ProMilk, IMM-124E, and Travelan as described in the legend to Fig. 1. Data represent mean ELISA units (EU) (plus SD where applicable) of antigen-specific IgG per milligram of total IgG. Values that are statistically significantly different between HBC and ProMilk tablets by two-sided two-sample t test are indicated by asterisks as follows: *, P < 0.05; ***, P < 0.01.

FIG 5

BCP inhibition of ETEC motility. Bacteria were seeded at the center of wells containing CFA agar mixed with serum or BCP. Migration was determined as the distance of growth after 4 to 6 h of incubation, and percent inhibition of motility was calculated with respect to migration in no serum or ProMilk. (A) The motility assay was optimized with immune sera (at the indicated dilutions) from volunteers challenged with strain H10407. Asterisks indicate significant differences between inhibition of motility in the presence and absence of serum. (B to D) ProMilk, IMM-124E, and Travelan were tested for their capacity to inhibit ETEC motility. (B and C) Inhibition of strain H10407 motility (B) and inhibition of strain E11881A, E1392/75/2A, and E9034 motility (C). Data shown in panels A to C represent mean percent inhibition of motility plus SD from at least three experiments; values that are significantly different between HBC and ProMilk by two-sided t test are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Representative images of zones of motility of ETEC strains (white bars) in agar containing ProMilk and IMM-124E.

FIG 6

Complement-mediated colostrum bactericidal activity of BCP. ProMilk, IMM-124E, and Travelan were tested for the ability to mediate killing of strain H10407 in the presence of complement. (A) Serially diluted ProMilk, IMM-124E, and Travelan supernatants were incubated with strain H10407 in the presence of baby rabbit complement for 1 h at 37°C, and the remaining viable organisms were quantified. Endpoint titers were determined as the dilution at which 50% of bacteria were killed using Reed-Muench regression. Data represent mean bactericidal titer plus SD from three experiments. Values that are significantly different (P < 0.001) between HBC and ProMilk by paired t test or Fisher's combined probability test are indicated by asterisks. (B) Inhibition of bactericidal activity by ETEC O78. Increasing concentrations of O78 COPS were preincubated with BCP (diluted at 1:25) to capture O78-specific antibodies, and the complement killing assay was performed as described above. Data represent the percent bacteria killed with respect to bacteria and complement-only control from three experiments. Values that are significantly different (P < 0.001) in the presence of O78 COPS by Fisher's combined probability test are indicated by asterisks.